1. Field of the Invention
The present invention relates to uniformly-sized, porous, small, round, hydrophilic polymeric beads and methods of making the beads and to the use of the beads in separation of molecules and in various analytical and diagnostic techniques.
The invention is directed to particles and the methods by which they are made and does not include tracer tagging or biological and clinical application tagging aspects disclosed and claimed in copending U.S. patent application Ser. No. 177,017, filed Sept. 1,1971, by William J. Dreyer and now U.S. Pat. No. 3,853,987.
2. Description of the Prior Art
There is a need for small, stable, spherical particles which are bio-compatible, i.e., do not interact non-specifically with cells or other biological components and which contain functional groups to which specific proteins and other bio-chemical molecules can be covalently bonded by established chemical procedures. Poly HEMA, a polyhydroxyethylmethacrylate, also known under the name Hydrogel, has been shown to possess blood compatible properties and is also used in the manufacture of contact lenses. The hydroxyl groups can be activated by cyanogen bromide for covalent bonding of proteins and other chemicals containing amino groups to the polymeric latex. Methacrylic acid residues which impart a negative charge onto the particles tend to prevent non-specific binding to cell surfaces and to provide carboxyl groups to which a variety of bio-chemical molecules can be covalently bonded using the carbodiimide method. Cross-linking of the polymeric matrix is essential to maintain the stability and size of the particles in both aqueous solution and in organic solvents commonly used in the fixation and dehydration of biological specimens for light and electron microscopy.
Knowledge of the nature, number and distribution of specific receptors on cell surfaces is of central importance for an understanding of the molecular basis underlying such biological phenomena as cell-cell recognition in development, cell communication and regulation by hormones and chemical transmitters, and differences in normal and tumor cell surfaces. In previous studies, the localization of antigens and carbohydrate residues on the surface of cells, notably red blood cells and lymphocytes, has been determined by bonding antibodies or lectins to such macromolecules as ferritin, hemocyanin or peroxidase which have served as markers for transmission electron microscopy. With advances in high resolution scanning electron microscopy (SEM), however, the topographical distribution of molecular receptors on the surfaces of cell and tissue specimens can be readily determined by similar histochemical techniques using newly developed markers resolvable by SEM.
Recently commercially available polystyrene latex particles have been utilized as immunologic markers for use in the SEM technique. The surface of such polystyrene particles is hydrophobic and hence certain types of macromolecules such as antibodies are absorbed on the surface under carefully controlled conditions. However, such particles stick non-specifically to many surfaces and molecules and this seriously limits their broad application. These particles are uncharged and are not capable of any ionic or covalent bonding of protein and other biological molecules.
HEMA particles possess chemical groups suitable for covalent bonding. However, homopolymers of HEMA are generally too soft for formation of porous beads and conventional suspension polymerization techniques are found to form fairly large particles on the order of 40 to 60 microns. Red blood cells and lymphocytes have a size of the order of 8 to 10 microns and in order to bind to specific receptor sites the beads must be of an order of magnitude smaller than the biological cell.
Small, uniformly-sized, cross-linked, porous, polyacrylic beads will also find use as a low-cost, stable absorbent in separating and purifying organic and inorganic compounds including polymers. The beads will also find use in chromatographic separation, filtration and gel permeation and affinity chromatography.
The hydrophilic organic gels commonly used in chromatography are sparsely cross-linked xerogels with a high swellability capacity in the eluent. They are characterized by considerable capacity ratio. However, the mechanical strength of the particles in the swollen state rapidly decreases with decreasing density of the cross-links. Application of the eluent under pressure at the column inlet frequency leads to plugging. Therefore, these gels are not suitable for high-speed gel chromatography. A new hydrophilic packing for gel chromatography is needed exhibiting high mechanical and hydrolytic stability.